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Abstract

Background

An association has been observed between the catechol-O-methyltransferase (COMT) gene, the predominant means of catecholamine catabolism within the prefrontal cortex
(PFC), and neuropsychological task performance in healthy and schizophrenic adults.
Since several of the cognitive functions typically deficient in children with Attention
Deficit Hyperactivity Disorder (ADHD) are mediated by prefrontal dopamine (DA) mechanisms,
we investigated the relationship between a functional polymorphism of the COMT gene and neuropsychological task performance in these children.

Methods

The Val108/158 Met polymorphism of the COMT gene was genotyped in 118 children with ADHD (DSM-IV). The Wisconsin Card Sorting
Test (WCST), Tower of London (TOL), and Self-Ordered Pointing Task (SOPT) were employed
to evaluate executive functions. Neuropsychological task performance was compared
across genotype groups using analysis of variance.

Conclusions

Contrary to the observed association between WCST performance and the Val108/158 Met polymorphism of the COMT gene in both healthy and schizophrenic adults, this polymorphism does not appear to
modulate executive functions in children with ADHD.

Background

Attention Deficit Hyperactivity Disorder (ADHD) is a childhood psychiatric disorder
characterized by symptoms of inattention, impulsivity and motor hyperactivity afflicting
6–8% of school-aged children in North America [1,2]. Although ADHD is a disorder with complex and heterogeneous etiology, genetic factors
appear to play a significant role in predisposing and perpetuating the development
of the disorder as evidenced by twin [3,4], family [5-7], and adoption studies [8]. Association studies have implicated several susceptibility loci including a 40-base
pair (bp) allele of the Variable Number of Tandem Repeats (VNTR) polymorphism of the
SLC6A3 gene [9] and a 48-bp repeat polymorphism of the DRD4 gene [10]. Attempts to replicate these findings have met with modest success possibly owing
to the clinical heterogeneity characteristic of the disorder [11]. One method that may act to augment the strength of these associations would be to
identify endophenotypic intermediates conferring risk for the development of ADHD
rather than attempting to identify direct linkages between genetic variations and
the behavioural manifestation of the disorder.

Theories of dysregulated dopamine (DA) pathways in ADHD have been supported by the
efficacy of dopamine agonists in reducing the core symptoms of the disorder [12]. The mesocortical DA pathway appears to be integral to prefrontal cortex (PFC)-mediated
cognitive functioning, specifically working memory [13], through the enhancement of task-related neural activity via D1 receptor activation
[14]. Both PET [15] and SPECT [16] imaging studies support a neuromodulatory role for DA in the PFC during tasks of
executive function. In addition, administration of DA agonists to the rat PFC acts
to enhance working memory in these animals [17]. Consistent with this line of thinking, children with ADHD show deficits in performance
of tasks of executive function [summarized in a meta-analysis by Sergeant et al. (2002)]
[18] and significant improvement of performance under methylphenidate [19,20]. These findings have prompted the hypothesis that the overt symptoms of ADHD are
the manifestation of an underlying deficiency in a range of PFC-mediated cognitive
domains, including working memory, planning, and set shifting, collectively regarded
as executive function [21-23].

The hypothesized role of a dysfunctional mesocortical dopaminergic pathway in the
development of symptoms of ADHD has encouraged the investigation of candidate genes
involved in this pathway including SLC6A3 [9], DRD4 [10] and, more recently, the catechol-O-methyltransferase (COMT) gene [24]. The COMT, encoded by a gene located on chromosome 22q11, catalyzes the degradation of catecholamines,
most importantly DA [25]. A functional polymorphism of this gene, involving a substitution of Valine (Val) for Methionine (Met) at codon 108/158 (Val108/158 Met), results in a 4-fold variation in enzyme activity, with individuals homozygous for
either the Val or Met allele exhibiting either reduced or preserved levels of DA respectively [26]. Although the dopamine transporter (DAT) is the predominant means of DA termination
in most dopaminergic neurons [27], considerable evidence exists to suggest that the DAT may play a reduced role within
the PFC [28-32], where other clearance mechanisms may be implicated. Comparison of DA metabolite
levels within discrete brain loci in both rats [33] and monkeys [34], as well as the measurement of DA levels in COMT knock-out mice [35], suggest an important functional role for COMT in the PFC. If COMT is indeed inextricably linked to DA metabolism within the PFC, it is reasonable to
assume that variations in enzyme activity, as dictated by the Val108/158 Met polymorphism, may modulate the performance of tasks of executive functioning in healthy
individuals, as well as individuals with reduced PFC basal dopamine levels. In support
of this assumption, associations have been reported between the Val108/158 Met polymorphism and performance on the Wisconsin Card Sorting Test (WCST) in healthy
adults [36,37]. In adults with Schizophrenia, a disorder characterized by dopaminergic hypofrontality
[38], associations have also been observed between the COMT polymorphism and WCST performance [39-41]. Although one study reported an association between the COMT polymorphism and ADHD using a haplotype relative risk design [24], this study failed to investigate any indices of executive function and several other
studies failed to replicate this finding [3,42-44].

Given the putative role of COMT in DA metabolism within the PFC [33-35], we hypothesized that the Val108/158 Met polymorphism of the COMT gene will be associated with alterations in performance on tasks of executive function,
a behavioural index of PFC integrity and function [45]. Since dysfunctional DA neurotransmission [46] and deficient neuropsychological task performance [18] are both characteristic of children with ADHD, we further hypothesized that this
association would be evident within this particular clinical population. Specifically,
ADHD children expressing the high enzymatic activity Val allele (H), resulting in reduced PFC DA neurotransmission [26], will show more pronounced deficits in neuropsychological task performance than their
low enzymatic activity Met allele (L) counterparts. In order to test this hypothesis, we used three measures
of executive function: the WCST [47], a measure of set-shifting ability capable of differentiating between ADHD children
and controls [18] and associated with the COMT polymorphism in normal [36,37] and schizophrenic adults [39-41]; the Tower of London (TOL) [48], a measure of planning ability, which consistently differentiates ADHD children from
controls [18], and the Self-Ordered Pointing Task (SOPT) [49], a measure of working memory also capable of differentiating between ADHD children
and controls [18].

Methods

Subjects

118 children were recruited from the Disruptive Behaviour Disorders Program and the
children outpatient clinic at the Douglas Hospital. They were referred to these specialized
care facilities by school principals, community social workers, and paediatricians.

Inclusion criteria required children to be between the ages of 6 and 12 years of age,
meeting DSM-IV diagnosis criteria for ADHD [50]. Diagnosis of ADHD was based on a structured clinical interview of parents using
the DISC-IV (parental report) [51], school reports, teacher interviews, and clinical observation of the child. In the
majority of cases, mothers were the primary informants for the collection of clinical
information. Written reports from the child's school were also available in the majority
of cases. Parents completed the Child Behavioural Checklist (CBCL) [52], a scale that assesses a variety of behavioural domains, and the Conners' Global
Index for parents (CGI-P) [53]. Teachers also completed the Conners' Global Index (CGI-T) [54]. Assessments were made while children were free of medication. Exclusion criteria
included a history of mental retardation, with an IQ less than or equal to 70 as measured
by the WISC-III [55], and history of Tourette Syndrome, pervasive developmental disorder, psychosis or
any medical condition or impairment that may interfere with the child's ability to
complete the study.

Neurocognitive assessment

A comprehensive neuropsychological test battery assessing different aspects of the
central executive functions was administered to all children by trained research personnel.
All children were assessed subsequent to a one-week medication "wash-out" period.
Children were permitted to take breaks upon request and, in some cases, testing was
carried out over two sessions. On average, the testing procedure lasted 1.5 hours.
The research protocol was approved by the Research Ethics Board of the Douglas Hospital.
Parents were explained the study and provided written consent. Children were also
explained the study and gave their assent to participate as well.

Tests were selected according to their ability to tap into various performance domains
of executive function. We restricted the number of tests in each domain in order to
balance comprehensiveness with the co-operation of patients. Abstraction and concept
formation were evaluated by means of the WCST (perseverative errors) [47]. In this task, children are required to sort cards according to three different criteria
(colour, number, or shape of signs presented on cards). Feedback on whether the child
achieved a correct or incorrect match is given after each trial. The matching criterion
changes after ten consecutive correct matches and the child has to identify the new
matching criterion using the feedback (correct/incorrect) provided to them. Evidence
of the reliability and validity of the WCST with various normal and clinical populations
has been reported in several studies [18]. Planning capacity was evaluated using the TOL [48]. This test is used to assess planning and problem solving aspects of executive functioning.
The validity and reliability of the TOL has been reported in numerous studies [18]. Standardized administration and scoring procedures as well as normative data have
been developed for paediatric populations [56]. Visual Working Memory was evaluated using the abstract version of the SOPT [49]. In this task, series of matrices of 6, 8, 10, and 12 images are presented to the
child. The child is asked to select, by pointing, one different image on each page.
Errors occur when the child points to images previously selected on the preceding
pages. Each set is presented to the child three times. Successful performance on this
task involves working memory as well as planning and monitoring skills. Shue & Douglas
(1992) have reported significant differences in performance between ADHD children
and normal controls on the SOPT [57].

Molecular genetics

The Val108/158 Met polymorphism of the COMT gene was genotyped using a PCR based method as previously described [26]. The PCR was performed in a 25 μl total reaction volume containing 1X PCR buffer,
200 uM dNTPs, 200 ng of primers (5'-GCGATGGTGGCACTCCAAGC; 5'-TTGGAGAGGCTGAGGCTGAC),
1 unit of Taq DNA polymerase, and 100 ng of genomic DNA. PCR products were electrophoresed
on agarose-TAE gel along with 1 kb ad 100 bp DNA ladders, visualized under UV-light
and coded according to the length of the PCR product. Genotypes were called by two
independent and experienced technicians who were blind to all clinical data. No disconcordance
in any of the readings was noted. Children were stratified according to genotype only
after all neuropsychological task data was collected.

Statistical analyses

The Val108/158 Met polymorphism consists of both the low-activity Met (L) and high-activity Val (H) alleles. Subjects were stratified into three groups: two homozygous genotype groups
(LL, HH) and one heterozygous genotype group (HL).

A one-way analysis of variance (ANOVA) where genotype (LL, HL, HH) was the independent
variable and neuropsychological task performance (standardized WCST perseverative
error score, standardized TOL total item score) was the dependent variable was performed.
For the SOPT, no normalized scores are available and testing procedures involve several
levels of difficulty (4). We therefore used a two-way, repeated measure, mixed design
analysis of covariance (ANCOVA), where genotype and level of task difficulty were
the between and within subjects independent variables, respectively, neuropsychological
task performance (SOPT raw error score) was the dependent variable, and age was the
covariate. As the TOL also involves multiple levels of task difficulty (12), we repeated
the analysis for this test using the same statistical approach as that applied to
the SOPT. A one-way ANCOVA, where genotype was the independent variable and age was
the covariate, was performed on all other non-standardized measures of neuropsychological
task performance (WCST number of categories completed, WCST number of trials to first
category, TOL number of problems solved).

Results

Table 1 shows clinical and demographic information for the children stratified according
to genotype [n = 23 for LL (19.5%), n = 66 for HL (56.0%) and n = 29 for HH (24.5%)].
The three groups were similar with regard to age, average household income, severity
of behavioural problems as assessed by the CBCL, and mean number of inattention items,
mean number of hyperactivity items and distribution of ADHD subtypes according to
the DISC-IV. No significant differences existed between the groups in IQ as measured
by the WISC-III. Our sample was characterized by a high prevalence of comorbid disorders,
particularly oppositional defiant disorder and conduct disorder. The frequency of
these disorders was equally distributed between the genotype groups. The proportion
of subjects who had never received medication for ADHD within each genotype group
was also remarkably similar. Although a significant effect of gender was observed
between genotype groups (χ2 = 7.39; df = 2, p = 0.02), this result was treated as a type I error (false positive)
due to the absence of female subjects with the HH genotype and given the relative
lack of female representation across all genotype groups. However, given the previously
observed association between gender and several polymorphisms at the COMT loci [59], increasing the sample size to achieve a more comparable gender representation and
distribution would be a valuable revision to the present design.

Table 1. Demographic and clinical characteristics of children with ADHD separated according
to COMT genotype

The genotype distribution conformed to a Hardy-Weinberg equilibrium (χ2 = 0.42; df = 2, p = 0.81). 156 parents participated in the study and gave blood samples.
Among these parents, 76 were heterozygous (M = 43 and F = 33) and transmitted the
Val allele to their affected children in 28 occurrences, whereas this same allele was
not transmitted in 29 occurrences [χ2 = 0.02; df = 1, p > 0.05 (transmission disequilibrium)]. Conversely, parents transmitted
the Met allele to their affected children in 29 occurrences, whereas this same allele was
not transmitted in 28 occurrences [χ2 = 0.02; df = 1, p > 0.05 (transmission disequilibrium)]. In addition, results from
the QTDT revealed no evidence of linkage or within-family association between the
three quantitative phenotypes and the COMT gene.

Discussion

Previous studies have identified an association between the COMT polymorphism and a variety of indices reflecting executive control both in healthy
[36,37] and schizophrenic adults [39-41]. The COMT appears to be important to the regulation of dopamine metabolism within the PFC [33-35]. Since the PFC and dopamine pathways have been hypothesized to play an important
role in the pathogenesis of ADHD [9-11,60,61]), we conducted this study in an attempt to test whether the COMT Val108/158Met polymorphism, which is known to be associated with a significant change in the catabolic
capacity of this enzyme, modulates the risk for ADHD or various indices of executive
control. Contrary to our expectations and findings in both healthy [36,37] and schizophrenic adults [39-41], an association between the Val108/158 Met functional polymorphism of the COMT gene and neuropsychological task performance reflecting executive control was not
observed in children with ADHD. This result is consistent with the findings of a recent
case-control study conducted by Mills et al. (2004), which, to our knowledge, is the
only other study to investigate the relationship between the COMT Val108/158Met polymorphism and neuropsychological task performance in children with ADHD [62]. However, this study did not include the WCST, the measure responsible for producing
the most consistent results in the previous literature. In addition, we did not identify
a biased transmission of either of the two alleles from parents to affected offspring.

The absence of an association between the COMT Val108/158Met polymorphism and behavioral indices of executive function in children with ADHD may
be explained by the young age of the population of patients included in the present
study. Indeed it is possible that, due to age-related changes in the functional importance
of the COMT within the prefrontal cortex, this association is observable only in adults. This
possibility is supported by data in both rats [63-65] and humans [66,67] suggesting that monoamine content and metabolism decrease with age. This age-related
decrease may render functions dependent on monoamine content more prone to be dysfunctional
at an older age. In addition, evidence from rat studies has indicated a positive correlation
between aging and COMT activity [68-70]. This observation may suggest that the implication of the COMT in the catabolism of dopamine is developmentally regulated, with children relying
less on this catabolic pathway than adults. Conversely, it has been reported that
DAT density is inversely correlated with age [71]. Taken together, the presence of an inverse and direct correlation between age and
DAT density on the one hand and COMT activity on the other hand, may suggest that dopamine metabolism relies more on the
DAT than on COMT activity in children compared to adults. This hypothesis is compatible with the fact
that several studies have identified an association between the DAT [9,60,72-74], but not the COMT, gene and ADHD.

It is also possible that the negative result observed in the present study is due
to a type II error (false negative) secondary to the lack of power of our sample to
detect an association. However, using results from the WCST, the variable for which
relevant genetic data already exists, we conducted a power analysis and determined
that our sample size has sufficient power (80% at α = .05) to detect a mean difference
of 11.2 on this measure. Furthermore, it is possible that some of the tests used in
our assessment are mediated by the PFC but insensitive to PFC DA levels [75].

An additional limitation of the present study is that some genotype groups included
few subjects. Increasing the sample size to achieve larger genotype groups would be
necessary to reach firmer conclusions. This is particularly true for female subjects
who were significantly underrepresented in the study (as is common to most clinical
studies of ADHD). In order to generalize these negative results to females, a more
comparable gender representation is required, particularly in view of some previous
research indicating that the allelic distribution of the COMT may be gender dependent
[59].

Conclusions

This study does not support the involvement of the Val108/158 Met polymorphism of the COMT gene in increasing the risk for ADHD or in modulating several indices of executive
functions in children with ADHD. This result is contrary to previous findings in both
healthy and schizophrenic adults and may be related to developmental specificities.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

ET performed the data analysis and drafted the manuscript. NG was involved in the
conception of the study and provided clinical support. LBA provided clinical support
and aided in data collection. PL provided clinical support. VM aided in neuropsychological
testing and data collection. RD and ATZ performed the genotyping for the study and
aided in data management. MTS coordinated the clinical aspects of the study and was
involved in data management. CB provided clinical support. RJ was responsible for
the conception of the study, drafting of the manuscript, and supervision of the research
project.

Acknowledgements

This work was supported in part by grants from the Fonds de la Recherche en Santé
du Québec, Réseau de Santé Mentale du Québec, and the Canadian Institutes of Health
Research to RJ and ET. We thank Johanne Bellingham, Anna Polotskaia and Nicole Pawliuk
for technical assistance.

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